Although the importance of protein histidine phosphorylation in mammals has been a subject of increasing interest, few chemical probes are available for monitoring and manipulating PHP activity. Here, we present an optimized and validated protocol for assaying the activity of PHPT1 using the fluorogenic substrate DiFMUP. The kinetic parameters of our optimized assay are significantly improved as compared with other PHPT1 assays in the literature, with a k of 0.39 ± 0.02 s, a K of 220 ± 30 μM, and a k/ K of 1800 ± 200 M s. In addition, the assay is significantly more sensitive as a result of using a fluorescent probe, requiring only 109 nM enzyme as compared with 2.4 μM as required by previously published assays. In the process of assay optimization, we discovered that PHPT1 is sensitive to a reducing environment and inhibited by transition-metal ions, with one apparent Cu(II) binding site with IC value of 500 ± 20 μM and two apparent Zn(II) binding sites with IC values of 25 ± 1 and 490 ± 20 μM.

Summary. Objective: Structural similarity between apolipoprotein(a) [apo(a)], the unique apoprotein of lipoprotein(a), and plasminogen (Plg), the zymogen for plasmin, results in inhibition of functions of Plg by apo(a) in vitro. The objective of this study was to evaluate the interaction of Plg and apo(a) in vivo. Methods and results: Vascular injury was induced in the carotid artery with a perivascular cuff in: (i) wildtype (WT); (ii) Plg deficient (Plg)/)); (iii) apo(a) (6 KIV construct) transgenic [apo(a)tg]; and (iv) apo(a) transgenic and Plg deficient [apo(a):Plg)/)] mice. At 10 days after cuff placement, the media and adventitia area were increased in the injured carotids compared with the uninjured carotids, and collagen deposition was greater in apo(a)tg, Plg)/) and apo(a):Plg)/) mice compared with WT mice. The incidence of a thrombus was greater (P < 0.05) in apo(a):Plg)/) mice (83%) than WT (20%), Plg)/) (12%), and apo(a)tg mice (9%). In the thrombi from apo(a)tg and apo(a):Plg)/) mice, P-selectin and von Willebrand factor immunostaining, indicating a platelet-rich thrombi, was greater than in WT and Plg)/) mice. The presence of fibrin(ogen) in the thrombi was greater in Plg)/) and apo(a):Plg)/) mice than apo(a)tg and WT mice. Of the four genotypes, only the apo(a):Plg)/) mice had both increased platelet and increased fibrin(ogen) deposition. Conclusions: The major finding of this study is the high incidence of thrombosis after vascular injury in apo(a)transgenic mice in a Plg deficient background, providing strong evidence for a prothrombotic role of apo(a) independent of Plg in vivo.

The protein tyrosine phosphatase (PTP) family of enzymes includes many attractive therapeutic targets, such as those in the leukocyte common antigen-related (LAR) subfamily of receptor PTPs. Synthesis and PTP inhibitory activity of illudalic acid and its methyl ether are described, with a focus on selective inhibition of LAR PTP relative to a small collection of other representative PTPs. The synthesis comprises 16 steps and provides illudalic acid in up to 12% overall yield from neopentylene-fused benzoate 1 (20 steps from commercial materials). Illudalic acid dose-dependently (measured IC 50 = 2.1 ± 0.2 μM) and time-dependently inhibits LAR consistent with previous reports of covalent binding. The kinetics of LAR inhibition by illudalic acid are consistent with a two-step mechanism in which the inhibitor and enzyme first interact noncovalently (K I = 130 ± 50 μM), followed by covalent ligation at a rate k inact = 1.3 ± 0.4 min −1 . The k inact /K I ratio of 10 4 corresponds to a t ∞ 1/2 of 0.5 min, as discussed herein. The phenol methyl ether of illudalic acid was found to be less potent in our dose−response assays (measured IC 50 = 55 ± 6 μM) but more selective for LAR, with a weaker initial noncovalent interaction and faster covalent ligation of LAR as compared to illudalic acid itself. A truncated analogue of illudalic acid that lacks the neopentylene ring fusion was found to be devoid of significant activity under our assay conditions, in contrast to previous reports. These observations collectively help inform further development of illudalic acid analogues as potent and selective inhibitors of the LAR subfamily of tyrosine phosphatases.

Two resorufin-based substrates for protein tyrosine phosphatase (PTP) activity have been synthesized. These substrates provide sensitive fluorogenic readouts of PTP activity in vitro and in living cells at both acidic and neutral pH. In addition, the presence of the pathogenic bacteria Staphylococcus aureus was detected visually using a colorimetric readout.